Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA...

Research ArticleCorrelation of Somatostatin Receptor-2Expression with Gallium-68-DOTA-TATE Uptake inNeuroblastoma Xenograft Models

Libo Zhang1 Douglass C Vines2 Deborah A Scollard2 Trevor McKee2 Teesha Komal2

Milan Ganguly2 Trevor Do2 Bing Wu1 Natasha Alexander1 Reza Vali1 Amer Shammas1

Travis Besanger3 and Sylvain Baruchel1

1The Hospital for Sick Children Toronto ON Canada2The STTARR Innovation Centre University Health Network Toronto ON Canada3The Center for Probe Development and Commercialization Hamilton ON Canada

Correspondence should be addressed to Sylvain Baruchel sylvainbaruchelsickkidsca

Received 1 March 2017 Revised 18 June 2017 Accepted 9 July 2017 Published 8 August 2017

Academic Editor Xuelei Ma

Copyright copy 2017 Libo Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATEhave become an effective treatment option for SSTR-positive neuroendocrine tumors The purpose of this study was to evaluatethe correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy inneuroblastoma (NB) xenograft models We demonstrated variable SSTR2 expression profiles in eight NB cell lines From micro-PET imaging and autoradiography a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts(CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)) Combined autoradiography-immunohistochemistryrevealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors With a low dose of 177Lu-DOTA-TATE (20MBqanimal) tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograftmodel Althoughin vitro NB cells showed variable expression levels of norepinephrine transporter (NET) a molecular target for 131I-MIBG therapylow 123I-MIBG uptake was observed in all selected NB xenografts In conclusion SSTR2 expression levels are associated with68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECTimaging in detecting NB tumors in our model Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging topotentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy

1 Introduction

Neuroblastoma (NB) is the most common extracranial child-hoodmalignancy responsible for 15 of all childhood cancerdeaths [1] Despite intensive treatment protocols includingmultimodal therapy with hematopoietic stem cell transplan-tation and immunotherapy three-year disease-free survivalis only about 60 for metastatic disease compared to 95 forlocalized tumors [2 3]

Somatostatin receptors (SSTRs) are expressed at relativelylow levels in most organs They are moderately expressed inthe brain gastrointestinal tract pancreas kidney and spleen

In contrast SSTRs especially SSTR2 have been shown tobe highly expressed in various human tumors includingpancreatic small cell lung and carcinoid tumors as wellas paraganglioma pheochromocytoma and neuroblastoma[4] Georgantzi et al demonstrated variable frequencies ofsomatostatin receptor (SSTR1-5) expression in 5 NB cell linesand 11 NB patient tumor biopsy samples [5] making molec-ular imaging and radionuclide therapy with somatostatin-based nuclear probes an attractive therapeutic option inappropriately selected patient populations [6]

DOTA-TATE is the somatostatin (cyclic peptide hor-mone) analog of Tyr3-octreotate (TATE) coupled with the

HindawiContrast Media amp Molecular ImagingVolume 2017 Article ID 9481276 10 pageshttpsdoiorg10115520179481276

2 Contrast Media amp Molecular Imaging

macrocyclic chelator 14710-tetraazacyclododecane14710-tetraacetic acid (DOTA) DOTA-TATE is SSTR2 selectivewith a higher SSTR2 affinity (sim02 nM) in vitro comparingto two other commonly used somatostatin analogs [68Ga-DOTA0-Tyr3]octreotide (DOTA-TOC) and [DOTA01NaI3]octreotide (DOTA-NOC) [7] Peptide-receptor imag-ing and therapy with radiolabeled somatostatin analogsare an established and effective treatment option for adultpatients with SSTR-positive neuroendocrine tumors [4]DOTA-TOC and DOTA-TATE can also be radiolabeled with90Y or 177Lu for targeted 120573minus-particle radionuclide therapyof neuroendocrine tumors In a recent phase I trial thesafety and efficacy of 90Y-DOTA-TOC therapy were demon-strated in 17 children and young adults with refractory SSTR-positive neuroendocrine tumors including NB [8] The firststudy of 177Lu-DOTA-TATE treatment in 35 patients withgastroenteropancreatic neuroendocrine tumors was pub-lished in 2003 where an objective response of 38 wasachieved [9] In a 2008 evaluation of 310 adult patientswith neuroendocrine tumors an overall response of 30was reported [10] More recently a pilot clinical studydemonstrated that 68Ga-DOTA-TATE PET could be used toimage children with relapsed or primary refractory high-riskNB and 68Ga-DOTA-TATE PET could be used to identifypotential candidates for 177Lu-DOTA-TATE treatment [11]In this study 6 out of 8 children demonstrated high uptakeof 68Ga-DOTA-TATE and proceeded to treatment Patientsreceived 2 or 3 administrations of 177Lu-DOTA-TATE(03GBqkg 81mCikg per dose) at a median interval of 9weeks and a median administered activity of 73 GBq Fiveof these patients had the stable disease as assessed using theResponse Evaluation Criteria in Solid Tumors (RECIST)This study while limited in the number of patients studiedprovided proof-of-principle that children with NB can beimaged and treated with somatostatin receptor-targetedagents More interestingly this study demonstrated that 1patient (out of a series of 6 patients) whose disease wasnegative for 123I-MIBG nevertheless demonstrated markeduptake of 68Ga-DOTA-TATE

The primary purpose of this study is to evaluate theuptake of 68Ga-DOTA-TATE in NB xenograft models andcorrelate this uptake with the expression levels of SSTR2and therefore identify biomarkers which can predict thetherapeutic effects of 177Lu-DOTA-TATE

2 Materials and Methods

21 Materials and Reagents Gallium-68 and lutetium-177radiolabeled DOTA-TATE were supplied by the Centre forProbe Development and Commercialization (CPDC Hamil-ton ON Canada) 68Ga-DOTA-TATEwas produced with thespecific activity of 412 plusmn 99GBq120583mol and with the radio-chemical purity of gt97 at all cases NET antibody (NET17-1) was purchased from MAb Technologies (Stone MountainGA) and SSTR2 antibody (ab134152) was obtained fromAbcam (Cambridge MA) Triton X-100 ethylenediaminete-traacetic acid (EDTA) and sodium dodecyl sulfate (SDS)

were purchased from Sigma Chemical Company (St LouisMO)

22 Cells and Cell Culture Eight NB cell lines (NUB-7 SK-N-BE(2) BE(2)C LAN-5 SH-SY5Y CHLA-15 CHLA-20and CHLA-90) were selected to represent a panel of celllines with different biological and genetic backgrounds ofNB (Table 1) NUB-7 LAN-5 SK-N-BE(2) BE(2)C andSH-SY5Y neuroblastoma cells were kindly provided by DrHerman Yeger (The Hospital for Sick Children TorontoON Canada) CHLA-15 CHLA-20 and CHLA-90 wereobtained from the Childrenrsquos Oncology Group Cell Cultureand Xenograft Repository (httpwwwcogcellorg) under asigned and approved Material Transfer Agreement Cell lineauthentication was performed using short tandem repeats(STR) DNA profiling (Promegarsquos GenePrint 10 System) [12]conducted by the Genetic Analysis Facility at the Centrefor Applied Genomics of The Hospital for Sick ChildrenThe DNA (STR) profile for all cell lines was matched tothe profile listed in the Childrenrsquos Oncology Group (COG)STR Database (httpstrdbcogcellorg) CHLA-15 CHLA-20 and CHLA-90 neuroblastoma cells were cultured inIscoversquos modified Dulbeccorsquos medium supplemented with3mML-glutamine 5120583gmL insulin 5120583gmL transferrin and5 ngmL selenous acid (ITS Culture Supplement Collabo-rative Biomedical Products Bedford MA) and 20 fetalbovine serum (FBS) NUB-7 LAN-5 SK-N-BE(2) BE(2)Cand SH-SY5Y neuroblastoma cells were cultured in 120572-MEMsupplemented with 10 FBS

23 RT-PCR Total cellular RNA was prepared using Qia-gen RNeasy mini kit (Qiagen Valencia CA) according tothe manufacturersrsquo instruction Residual DNA was elim-inated using the Qiagen RNase-Free DNase Set cDNAswere synthesized from 2 120583g of RNA with the SuperscriptII Reverse Transcriptase (Invitrogen) PCR was performedusing 1120583L of cDNA in the PCR buffer supplemented with02mM of dNTP 25 units of Taq polymerase (Biorad andThermoFisher Scientific) and 05 120583M of each sense andantisense primer The following primers were used SSTR2forward 51015840-GGTGAAGTCCTCTGGAATCC-31015840 and reverse51015840-CCATTGCCAGTAGACAGAGC-31015840 NET forward 51015840-CTCAAGGAGGCCACGGTATGGATCG-31015840 and reverse 51015840-ACCTGGAAGTCATCAGCCAGTCCGG-31015840 GAPDH for-ward 51015840-CTGTCCAGTTAATTTCTGACC-31015840 and reverse 51015840-CTTTGTACATGGTATTCACCAC-31015840 PCR products wererun on a 15 agarose (Invitrogen) with a 100 bp marker(Thermo Fisher Scientific) and stained with ethidium bro-mide Gel pictures were taken using the AlphaImager 2200(Alpha Innotech Kasendorf Germany)

24 Western Blot Theprotein lysates were analyzed byWest-ern blot for SSTR2 and norepinephrine transporter (NET)Briefly cells were lysed in lysis buffer and denatured Sampleswere separated using 10 Bis-Tris precast gels (Invitrogen)followed by transfer to a PDVFmembrane After blocking allmembranes were incubated overnight at 4∘C in TBST (Tris-buffered saline 01Tween 20) buffer containing the primaryantibodies Primary antibody complexes were then detected

Contrast Media amp Molecular Imaging 3

Table 1 Neuroblastoma cell lines used in this study

Cell line Site Stage Patient age Phase of therapy MYCN amp p53 mutantCHLA-15 Tumor 4 gt1 DX N WTCHLA-20 Tumor 4 15 PD-Ind N WTCHLA-90 BM 4 85 PD-Auto-BMT N MutLAN-5 BM Unknown 04 Unknown A WTNUB-7 LN 4s4 07 Unknown A WTSH-SY5Y BM 4 4 PD-Ind N WTBE(2)C BM 4 22 PD-Ind A MutSK-N-BE(2) BM 4 22 PD-Ind A MutBM= bonemarrow B = bone L = liver P = pulmonary LN = lymph node DX = at diagnosis PD-Ind = progressive disease on induction chemotherapy BMT= bone marrow transplantation PD-Auto-BMT = relapsed after myeloablative chemo-radiotherapy followed by bone marrow transplantationWT =wild typeMut = mutant N = nonamplified A = amplified

using horseradish peroxidase- (HRP-) conjugated secondaryantibodies Protein bands were revealed with SuperSignalWest Pico Chemiluminescent Substrate (Thermo Fisher Sci-entific) NET protein expression in CHLA-15 SK-N-BE(2)and BE(2)C xenografts was quantified densitometricallyusing ImageJ software (NIH USA) and normalized withrespect to the corresponding expression of 120573-actin

25 Tissue Preparation for Western Blot Xenograft tumorswere snap frozen in liquid nitrogen immediately after har-vesting and stored at minus80∘C until ready for processingTumor tissue samples were homogenized in a RIPA buffer(150mM NaCl 50mM Tris-HCl 05mM EDTA 1 TritonX-100 and 01 SDS) plus complete protease inhibitorcocktail (Complete Protease Inhibitor Tablets BoehringerMannheim Ingelheim am Rhein Germany) Homogenateswere then centrifuged at 100000timesg for 45 minutes at 4∘CThe supernatants were assayed for protein content aliquotedand stored at minus80∘C 25120583g of lysate was subjected to futureprotein analysis by SDS-PAGE and Western blot analysis

26 Mouse Xenograft Models All animal studies were ap-proved by Animal Care Committee at the Hospital for SickChildren and at theUniversityHealthNetwork (TorontoONCanada) Four- to 6-week-old female nonobese diabeticsevere combined immunodeficiency (NODSCID)micewerepurchased from Jackson Laboratory (Bar Harbor ME)CHLA-15 SK-N-BE(2) and BE(2)C cells were used to estab-lish murine models Briefly tumor cells were washed threetimes with Hanksrsquo Balanced Salt Solution (HBSS) beforeinjection Cell suspensions were mixed 1 1 with Growth Fac-tor Reduced Matrigel Matrix (BD Bioscience MississaugaON Canada) Subcutaneous xenografts were developed byinjecting 1 times 106 tumor cells subcutaneously into the dorsalupper flank of NODSCID mice

27 Micro-PETCT Mouse Imaging Animals were preparedfor imaging such that when the tumor xenografts reach adiameter of approximately 1 cm the mice were anesthetizedwith 2 isoflurane in the medical air (10 Lmin) and injectedintravenously (IV) via the tail vein [13] with 117MBq plusmn25MBq of 68Ga-DOTA-TATE Images were acquired on a

Focus 220 micro-PET scanner (Siemens Preclinical Solu-tions Knoxville TN) at 1 hour after injection using aMinerve imaging bed (Esternay France) to maintain bodytemperature at 37∘C Images were reconstructed in a 256 times256matrix and a zoomof 65 using an ordered subset expecta-tion maximization (OSEM) followed by a maximum a pos-teriori probability reconstruction algorithm with no attenu-ation correction Quantification was performed by volume-of-interest (VOI) analysis using Inveon Research Workplace(IRW) software (Siemens) Tumor volume was obtained bysumming multiple 2-dimensional regions of interest fromconsecutive tomographic planes encompassing the entiretumor volume on fused PET-CT slices Tumor uptake wasexpressed as the mean plusmn SD percentage injected dose pergram (IDg)

Immediately after small-animal PET the Minerve imag-ing bed with the mouse was transferred to an eXplore LocusUltra Preclinical CT scanner (GE Healthcare London ONCanada) The micro-CT scan of the mouse was acquiredwith routine acquisition parameters (80 kV 70mA 16 secper rotation) PET and CT images were coregistered usingIRW software The micro-CT scan was used for anatomicreferencing and for delineating the aforementioned VOIs

To verify the accuracy of ID values a 10mL specimenof 68Ga with known radioactivity was scanned on the micro-PET The image-derived concentration was compared withthe concentration calculated from the same radioactivitymeasured by the radioisotope dose calibrator (Model CRC-15R Capintec Inc Ramsay NJ) The difference in the twoconcentrations was less than 10

28 123I-MIBG SPECTCT Imaging 1 times 106 NB tumorcells were injected subcutaneously into the shoulder areaof NODSCID mice When tumor xenografts grew to anapproximate diameter of 1 cm or more each mouse wasinjected with 175MBq plusmn 23MBq of 123I-MIBG into a lateraltail vein Five to six hours after injection both CT and SPECTimaging were performed using a preclinical nanoSPECTCT system (Bioscan Washington DC) For imaging micewere anesthetized with 15 isoflurane and medical air at10 Lmin For anatomical reference the cone-beam CT scanwas acquired first at 45 kVp and 177 120583A Image slices werereconstructed in a 176 times 176 matrix with a fast filtered


back-projection algorithm using InVivoScope 143 software(Bioscan USA)

For the SPECT scan a 20 window was set around the159 keV principal gamma-photon of I-123 In thismultiplexedmultipinhole SPECT system the 14mmnine-pinhole mouseldquostandardrdquo collimators were attached to each of the fourdetector heads consisting of NaI(Tl) crystals Photons wereacquired for about 150 sprojection and 24 projections perdetector head in a 256 times 256 matrix for a total imagingtime ranging from 60 to 75 minutes SPECT data werereconstructed by ordered subset expectation maximization(OSEM) methods with four subsets of data undergoing 9iterations each using InVivoScope 143 The CT and SPECTslices were then coregistered Image analysis and volume-of-interest (VOI) quantification were performed using Vivo-Quant 25 (MedisoinviCRO Boston MA) The activityconcentration in the VOI for the whole tumor was divided bythe activity concentration in theVOI for the hind limbmusclein order to calculate the tumor-to-muscle ratio (119879119872)

29 Autoradiography After the 68Ga-DOTA-TATE PETCTand 123I-MIBG SPECTCT studies CHLA-15 SK-N-BE(2)and BE(2)C xenografts were harvested cut in half embeddedin Tissue-Tek optimum cutting temperature (OCT) com-pound (Tissue-Tek Sakura Torrance CA) along with a pieceof forelimb muscle as control and frozen on liquid nitrogenvapor Frozen blocks were transferred to the STTARR cor-relative pathology lab on dry ice and serial frozen sectionsof alternating 5 120583m (for immunohistochemistry) and 50120583mthickness (for autoradiography) were cut placed on glassslides and left to dry for 20 minutes After sections werecompletely dry the 50 120583m sections were placed in a custom-built 16-slide holder that held the frozen sections in closeproximity to a storage phosphor screen (Cyclone Plus StoragePhosphor System Perkin Elmer Shelton CT USA) with alayer of plastic wrap separating them In some instances apiece of filter paper with serial dilutions of radiotracer wasincluded in the cassette for use as a standard and to check thelinearity of the film This cassette was maintained at minus20∘Cfor a period of time equating to 10 half-lives of activity Thetiming of tumor resection and contact with phosphor screenwere recorded for all cases Following completion of 10 half-lives of decay the phosphor screen was removed from thecassette and developed on the Cyclone Plus imaging systemat 600 dpi resolution with a written record of slide locationson the screen Image quantification was performed in ImageJsoftware in which regions of interest (ROIs) were drawnaround each tumor and corresponding piece ofmuscle tissueand mean phosphor intensity was recorded in each region AROI corresponding to background signal was also recordedtaken from the region of screen not containing any slidesor tissue Mean tumor-to-muscle ratios were calculated bydivision of mean per-pixel intensity in tumor (subtractingbackground) divided by mean per-pixel muscle intensitysubtracting background

210 SSTR2 Immunohistochemistry SSTR2 immunofluores-cence staining was performed on 5 120583m thick frozen sec-tions Tissue sections were fixed for 10 minutes in acetone

and allowed to air dry for 5 minutes Endogenous biotinbiotin receptors and avidin sites were blocked with theAvidinBiotin Blocking Kit (SP-2001 Vector LaboratoriesBurlingame CA) The tissue sections were incubated withrabbit anti-SSTR2 antibody (1 100 ab9550 Abcam) for 1hour at room temperature Detection of the rabbit antibodieswas performed by incubation with Texas Red goat anti-rabbit IgG antibody (1 200 TI-1000 Vector Laboratories)for 30 minutes For the positive control of SSTR immuno-histochemistry normal pancreas islets were used Negativecontrols were done by omitting the specific primary anti-bodies and processed in the same way The tissue sectionswere washed and then mounted with Vectashield mountingmedium with DAPI (H-1200 Vector Laboratories) Whole-slide scanned immunofluorescence images were acquired ona TissueScope 4000 (Huron Technologies Waterloo ONCanada) at 1 120583mpixel resolution Coregistration of auto-radiography with immunofluorescence was performed byupsampling the autoradiography image (600 dpi whichequates to 42 120583mpixel resolution) of the 50 120583m adjacentsection to match the immunofluorescence image (imaged at1 120583mpixel resolution) followed by rigid registration using acustomized MATLAB script

211 In Vivo Treatment with 177Lu-DOTA-TATE Drug treat-ment commenced when the tumor sizes reached 05 cm indiameter Animals were randomized into two groups eachwith 7 animals the control group and the 177Lu-DOTA-TATE treatment group A single dose of 177Lu-DOTA-TATE(20MBq) [14] was administered as treatment Control micereceived the same volume of saline Tumor growth wasmonitored by measuring tumor dimensions using a digitalcaliper Tumor volumewas calculated as width2times lengthtimes05When tumor volume reached 3 cm3 mice were sacrificedand tumors were dissected and weighed Tumor growthcurves consisting of the tumor volumes at different timepoints were plottedDuring the study themicewere observeddaily for possible adverse effects due to treatmentsMorbiditysigns of ill health such as ruffledthinning fur abnormalbehaviors or local erosion from the tumor were observedAnimal body weight was also monitored for general toxicity

212 Statistical Analysis Data from different experimentswere presented as mean plusmn SD Two-tailed unpaired Studentrsquos119905-tests were performed to compare the uptake values obtainedfrom SPECTCT imaging and tumor growth in two differ-ent groups 119879119872 ratios of 123I-MIBG uptake and of 123I-MIBG autoradiography in different groups were analyzedby one-way analysis of variance (ANOVA) and Tukeyrsquos testComparison of NET expression between different groupswas analyzed by the nonparametric Kruskal-Wallis test withDunnrsquos multiple comparison tests Statistical significance wasachieved with a two-sided 119875 lt 005 All statistics weregenerated using GraphPad Prism software version 6

3 Results

31 Variable Expression Levels of SSTR2 and NET inNeuroblastoma (NB) Tumor Cell Lines The expression of


SSTR2

NET

GAPDH

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(a)

SSTR2

NET

-Actin

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(b)

Figure 1 mRNA expression level and Western blotting analyses of SSTR2 and NET (a) RNA was isolated from different neuroblastoma celllines converted into cDNA followed by RT-PCR with SSTR2 and NET specific primers The GAPDH gene was used as a reference gene (b)Protein lysates were prepared from different neuroblastoma cell lines Protein samples were separated by polyacrylamide gel electrophoresisExpression of SSTR2 and NET proteins was visualized using specific antibodies 120573-Actin was used as internal loading control

somatostatin receptors (SSTR2) and NET in NB cell lineswas determined using RT-PCR (Figure 1(a)) andWestern blot(Figure 1(b)) Although different NB cell lines showed similarSSTR2 mRNA expression levels (Figure 1(a)) marked varia-tion of SSTR2 protein expression was observed (Figure 1(b))In some NB cell lines such as CHLA-15 CHLA-20 CHLA-90 and LAN-5 a prominent SSTR2 expression was detectedwhereas in others a low level of SSTR2 expression wasidentified (Figure 1(b)) A similar variation was observed forthe expression of NET a primary transporter responsible forspecific active cellular uptake of MIBG [15] (Figures 1(a) and1(b)) Interestingly some high SSTR2-expressing cell linesCHLA-15 CHLA-90 and LAN-5 showed low expressionlevels of NET which makes SSTR2 a potential alternativemolecular target for NB imaging or treatment especially forMIBG nonacid tumors

In addition SSTR2 or NET expression did not appearto be correlated with MYCN amplification or p53 mutationstatus In some cell lines two bands were detected using themonoclonal SSTR2 antibody (ab134152 Abcam) which isconsistent with a previous report in which two bands werealso detected in IMR-32 neuroblastoma cell lysates [16]

32 Uptake of 68Ga-DOTA-TATE Correlates with SSTRT2Expression in NB Xenografts DOTA-TATE has a high affin-ity for SSTR2 in vitro In order to assess the relationshipbetween 68Ga-DOTA-TATE uptake and SSTR2 expressionwe selected a high SSTR2-expressing NB cell line CHLA-15 and a low SSTR2-expressing cell line SK-N-BE(2) for invivo PETCT tumor imaging Tumor uptake was expressedas Standardized Uptake Value (SUV) As shown in Figures2(a)ndash2(c) we observed a significant difference in the uptakeof 68Ga-DOTA-TATE between CHLA-15 and SK-N-BE(2)xenografts The mean tumor uptake value of 68Ga-DOTA-TATE was significantly higher in the CHLA-15 xenografts(079plusmn 010 IDg 119875 = 00003) compared to SK-N-BE(2)tumors (013plusmn 002 IDg 119875 lt 001)

33 Histological Colocalization of SSTR2 and 68Ga-DOTA-TATE Uptake To further evaluate the relationship betweenSSTR2 expression and 68Ga-DOTA-TATE uptake we per-formed SSTR2 immunostaining and ex vivo autoradiog-raphy with CHLA-15 and SK-N-BE(2) xenograft sectionsConsistent with the PET results CHLA-15 tumors showedsignificantly higher accumulation of 68Ga-DOTA-TATE ascompared to SK-N-BE(2) tumors (Figures 3(a) and 3(b))We also observed spatial heterogeneity for both SSTR2expression (Figure 3(c)) and 68Ga-DOTA-TATE accumula-tion (Figures 3(a) and 3(d)) When wemerged the SSTR2 flu-orescent staining and autoradiography images we observedintratumoral colocalization of SSTR2 expression and 68Ga-DOTA-TATE uptake (Figure 3(d)) Tumor regions with ahigh number of SSTR2-positive cells corresponded to focalareas of increased radioactivity In SK-N-BE(2) tumors bothSSTR2 expression and 68Ga-DOTA-TATE autoradiographysignals were too weak to be detected (Figure 3(e))

34 Therapeutic Effects of 177Lu-DOTA-TATE on the CHLA-15 Xenograft Model To verify the therapeutic effects oftargeting SSTR2 with 177Lu-DOTA-TATE we treated CHLA-15 tumor-bearing mice with 177Lu-DOTA-TATE at the doseof 20MBqanimal After 12 days we started to observe sig-nificant tumor growth inhibition in the CHLA-15 xenograftmodel compared to control tumors (119875 lt 005) (Figure 4(a))From the slope of tumor growth curve Lu-177-DOTA-TATEtreated tumors regained tumor growth rate after day 12which indicates that single dose Lu-177-DOTA-TATE maynot achieve long-lasting antitumor effects No overall toxicitywith respect to body weight loss was observed at the dose of20MBqanimal (Figure 4(b))

35 123I-MIBG Uptake by NB Xenografts Not Related toTheir NET Expression In Vitro To compare the effectivenessbetween 68Ga-DOTA-TATE PET and 123I-MIBG SPECT inour preclinicalmodels we further assessed 123I-MIBGuptake


68-Ga-DOTA-TATE uptake by CHLA-15 xenograft

(a)

68-Ga-DOTA-TATE uptake by SK-N-BE(2) xenograft

(b)10

08

06

04

02

00

Tum

or S

U６

Ｇ

Ｈ

lowastlowast

CHLA-15 SK-N-BE(2)

(c)

Figure 2 Representative micro-PETCT images at 1 hour after injection of 10MBq of 68Ga-DOTA-TATE in the CHLA-15 (a) and SK-N-BE(2) (b) tumor-bearing NODSCIDmice Images were presented in the axial (left) and coronal (right) orientationsThewhite arrows denotelocalized tumor on the shoulder (c) Standardized Uptake Values (SUV) in CHLA-15 and SK-N-BE(2) xenografts were calculated using theformula SUV = 119862PET(119879)(Injected doseBodyweight) The difference was significant between CHLA-5 and SK-N-BE(2) tumors (lowastlowast119875 lt001) Two-tailed unpaired 119905-tests were performed to compare the SUVmean values obtained

in three neuroblastoma xenograft models including NB cellsexpressed high intermediate and low amount of SSTR2protein in vitro BE(2)C SK-N-BE(2) and CHLA-15 cellsrespectively As shown by 123I-MIBG SPECT (Figures 5(a)and 5(b)) and 123I-MIBG autoradiography (Figure 5(c))uneven marginal uptake of 123I-MIBG was observed in allthree BE(2)C SK-N-BE(2) and CHLA-15 xenograft tumorsAlso in vivo 123I-MIBG uptake is not related to in vitroNETexpression levels in three selected NB cell lines AlthoughBE(2)C cells had a relatively higher NET expression in vitrotheir in vivo 123I-MIBG uptake is at the similar level as thebackground with the 119879119872 ratio of 113 plusmn 013 We werenot able to detect positive signals from NET immunohisto-chemistry staining with harvested NB xenografts In order tocompare the NET expression in vivo we ran aWestern blot ofNETwith homogenized tumor samples (Figure 5(d))We didnot however observe a significant difference of NET expres-sion between CHLA-15 SK-N-BE(2) and BE(2)C xenograftswhich indicates a possible change of NET expression profiledue to in vitro cell culture (Figure 5(e))

4 Discussion

Neuroblastoma is an extremely heterogeneous disease bothbiologically and clinically comprising tumor cells with verydifferent molecular features Somatostatin receptors (SSTRs)are variably expressed in neuroblastoma cell lines andtumors as demonstrated by autoradiography Western blotimmunohistochemistry and RT-PCR techniques [17ndash19] Inthis study we selected a panel of NBL cells lines withdifferent biological and genetic backgrounds (Table 1) Wedetected SSTR2 mRNA in most of the selected cell linesbut only 4 out of 8 cell lines express high levels of SSTR2protein includingCHLA-15 CHLA-20 CHLA-90 and LAN-5 cells In addition expression of SSTR2 was not relatedto the p53 mutation and MYCN amplification status Sincethe commercially available NB cell lines are mostly derivedfrom stage IV patient samples we are currently conductinga large-scale Childrenrsquos Oncology Group biological study(COG ANBL14B3) with both high-risk and non-high-riskNB patient specimens to investigate the relationship betweenSSTR2 expression and clinical features


SK-N-BE(2)

(b)

CHLA-15

(a)

Red SSTR2Blue DAPIWhite autoradiography

(c) (d) (e)

2mm

Figure 3 Colocalization of SSTR2 and autoradiography on CHLA-15 xenografts CHLA-15 and SK-N-BE(2) tumors were removedimmediately after PETCT imagingThe spatial distribution of the 68Ga-DOTA-TATEuptakewas visualized by autoradiography (white signal)in the serial sections of CHLA-15 (a) and SK-N-BE(2) (b) tumors (c) Representative CHLA-15 tumor section was stained for SSTR2 (redfluorescence) and DAPI (blue fluorescence) (d) The merging image of SSTR2 immunostaining and autoradiograph of the same CHLA-15tumor section (e) A representative SK-N-BE(2) tumor section stained for SSTR2 (red fluorescence) and DAPI (blue fluorescence)

0 10 15 200

1

2

3

4

Days after drug treatment5

Tum

or v

olum

e (c Ｇ

3)

Control177Ｏ-DOTA-TATE

lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t

0 10 15 20Days after drug treatment

5


(b)

Figure 4 Antitumoral effects of 177Lu-DOTA-TATE in the CHLA-15 neuroblastoma model (a) Mice (119899 = 7) with subcutaneous CHLA-15xenografts were treated with one dose of 20MBq of 177Lu-DOTA-TATE Control mice (119899 = 7) received saline Tumor volume was measuredand plotted as shown Values are stated as meanplusmn SE lowast119875 lt 005 (b) Animal body weight was monitored in CHLA-15 tumor-bearing micewithwithout 177Lu-DOTA-TATE treatment


SK-N-BE(2) CHLA-15 BE(2)C

(a)

0

2

4

6

8

10

SKNBE2 CHLA15 BE(2)C

）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast

）123-MIBG autoradiography

Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)

Figure 5 (a) Example images of 123I-MIBG SPECTCT in the SK-N-BE(2) CHLA-15 and BE(2)C xenograftmodelsThewhite arrows denotelocalized tumor on the shoulder (b) Scatter plots of the tumor-to-muscle (119879119872) ratios of 123I-MIBG uptake in the SK-N-BE(2) CHLA-15and BE(2)C xenograft models derived from SPECTCT imaging One-way analysis of variance (ANOVA) with Tukeyrsquos test was used forstatistical analysis lowast119875 lt005 lowastlowast119875 lt 001 (c) Scatter plots of the tumor-to-muscle (119879119872) ratio of 123I-MIBG autoradiography in the SK-N-BE(2) CHLA-15 and BE(2)C xenograftmodels One-way ANOVAwith Tukeyrsquos test was used for statistical analysis lowast119875 lt 005 lowastlowast119875 lt001 (d)Western Blot of NET with homogenized CHLA-15 SK-N-BE(2) and BE(2)C xenografts Three tumors were randomly selected from threegroups forWestern blot analysis (e) Quantitative analysis of NET protein expression in CHLA-15 SK-N-BE(2) and BE(2)C xenografts NETprotein expression in Western blot images was quantified densitometrically using ImageJ software (NIH USA) and normalized with respectto the corresponding expression of 120573-actin Comparison of NET expression between different groups was analyzed by the nonparametricKruskal-Wallis analysis with Dunnrsquos multiple comparison tests No significant difference of NET expression was observed between CHLA-15SK-N-BE(2) and BE(2)C xenografts


Peptide-receptor imaging and therapy with radiolabeledsomatostatin analogs are an established and effective treat-ment option for adult patients with SSTR-positive neuro-endocrine tumors [4] 111In-[diethylenetriaminepentaaceticacid (DTPA)]octreotide (OctreoscanMallinckrodt) a soma-tostatin analog has been used for more than 15 years in thediagnosis and staging of SSTR-positive tumors Neverthelessit has a restricted ability to identify lesions smaller than 1 cmand to obtain a good spatial resolution even when usingSPECT rather than planar imaging [20] In recent yearsnew PET-based radiopharmaceuticals targeting somato-statin receptors have been developed to address these issuesSeveral studies have demonstrated that 68Ga-labeled DOTA-TOC or DOTA-TATE PET combined with CT has distinctlyhigher sensitivity and improved spatial resolution for thedetection of SSTR-positive neuroendocrine tumors com-pared to scintigraphy with conventional SPECT imagingusingOctreoscan [4 21] In this study we successfully imaged68Ga-DOTA-TATE uptake in SSTR2-positive neuroblastomaxenografts with micro-PETCT We also observed intensive68Ga-DOTA-TATE uptake in MIBG-low-avidity CHLA-15xenografts131I-MIBG has been used over the past 15 years in

multimodal therapy as a radiotherapeutic agent in relapsedand refractory NB patients [22] However only 30ndash40 ofchildren with chemotherapy-refractory disease respond to131I-MIBG and the responses are usually only transient [22ndash24] In this study we observed low 131I-MIBG avidity in allselected NB cell lines Although BE(2)C cells showed rela-tively elevated NET expression in vitro we were not able todetect positive NET immunostaining with harvested BE(2)Cxenografts Western blot of NET with homogenized tumorsamples showed similar NET expression levels betweenCHLA-15 SK-N-BE(2) and BE(2)C xenografts which indi-cates a possible change of NET expression profile in vivo Wealso demonstrated the complementary role of somatostatinreceptor imaging in detecting additional sites of neuroen-docrine tumors that were not visualized with 123I-MIBGscintigraphy Several clinical studies also confirmed discor-dant uptake patterns of MIBG and somatostatin receptorexpression in some NB tumors [25] Kroiss et al [26] forexample described that somatostatin receptor imaging with68Ga-DOTA-TOC PET was able to detect the sites of diseasein 24 patients with NB which were not visible by 123I-MIBGNumerous other studies in patients with neuroendocrinetumors have similarly demonstrated the complementary roleof somatostatin receptor imaging in detecting additional sitesof disease that were not visualized with 123I-MIBG scintigra-phy [27] In this study we observed discordant expression ofNET and SSTR2 expression in NB-cell lines CHLA-15 has alower level of NET expression but higher SSTR2 expressioncompared to SK-N-BE(2) cells In vivo PETCT imagingdemonstrated high 68Ga-DOTA-TATE uptake in CHLA-15xenografts compared to SK-N-BE(2) tumors Consequentlysomatostatin receptor expression and DOTA-TATE imagingcould serve as an adjunct to existing diagnostic and thera-peutic methods They may provide valuable information for

the pretherapeutic staging of the disease and impact patientoutcomes

One of the major goals of our study is to understand howthe molecular expression of SSTR2 determined the responsesof radioactively labeled DOTA-TATE DOTA-TATE can alsobe radiolabeled with 177Lu for targeted 120573minus-particle radio-therapy 177Lu has a similar physical half-life as 131I 177Luemits a medium-energy 120573minus-particle resulting in localizedenergy deposition thus the targeted tissue receives low-dose-rate radiation exposure Mouse studies of solid tumorxenografts found 177Lu to be superior to other radiolan-thanides in effecting tumor control with pretargeted therapy[28] We demonstrated the antitumor effect of 177Lu-DOTA-TATE on CHLA-15 xenografts which have high SSTR2expression and elevated uptake of 68Ga-DOTA-TATE Thesecharacteristics may be valuable in future clinical trials both68Ga-DOTA-TATE PET imaging and SSTR2 protein profilecould serve as indicators for 177Lu-DOTA-TATE treatmentresponseThis avenue needs to be evaluated further in clinicalscenarios

5 Conclusions

This study has allowed us to demonstrate the associationbetween SSTR2 expression and 68Ga-DOTA-TATE uptakewhich potentially leads to the antitumor activity of 177Lu-DOTA-TATE in NB preclinical models Histological colocal-ization of SSTR2 and 68Ga-DOTA-TATE was also observedin our study SSTR2 expression therefore could be used as apotential biomarker for predicting drug response to 177Lu-DOTA-TATE radiotherapy Moreover in our model wedemonstrated that 68Ga-DOTA-TATEPET is superior to 123I-MIBG SPECT imaging in detecting NB xenograft tumorsThe absence of significant difference of NET expressionbetween various NB xenografts models and discordant invitro and in vivoNET expression represent a limitation of ourstudy which will require further investigation

The ongoing COG study looking at the prevalence ofSSTR2NET expression in high-risk NB patients may allowus to identify a subset of patients who could benefit from thisnew SSRT2 targeted radiotherapeutic modality in particularfor the small number of patients demonstrating MIBG-nonavid tumors

Conflicts of Interest

No potential conflicts of interest were disclosed by theauthors

Acknowledgments

This work was supported by CJ Memorial Fund NuMedKidsFund SickKids Foundation and JamesBirrellNeuroblastomaResearch Fund The authors would like to acknowledge theSpatio-Temporal Targeting and Amplification of RadiationResponse (STTARR) program and its affiliated funding agen-cies


References

[1] R Howman-Giles P J Shaw R F Uren and D K V ChungldquoNeuroblastoma andOtherNeuroendocrine Tumorsrdquo Seminarsin Nuclear Medicine vol 37 no 4 pp 286ndash302 2007

[2] H Mugishima ldquoCurrent status of molecular biology and treat-ment strategy for neuroblastomardquo International Journal of Clini-cal Oncology vol 17 no 3 p 189 2012

[3] I Ora and A Eggert ldquoProgress in treatment and risk stratifi-cation of neuroblastoma impact on future clinical and basicresearchrdquo Seminars in Cancer Biology vol 21 no 4 pp 217ndash2282011

[4] H RMaecke and J C Reubi ldquoSomatostatin receptors as targetsfor nuclear medicine imaging and radionuclide treatmentrdquoJournal of Nuclear Medicine vol 52 no 6 pp 841ndash844 2011

[5] K Georgantzi A V Tsolakis M Stridsberg A Jakobson RChristofferson and E T Janson ldquoDifferentiated expression ofsomatostatin receptor subtypes in experimental models andclinical neuroblastomardquo Pediatric Blood and Cancer vol 56 no4 pp 584ndash589 2011

[6] F D Pashankar M S OrsquoDorisio and Y Menda ldquoMIBG andsomatostatin receptor analogs in children current concepts ondiagnostic and therapeutic userdquo Journal of Nuclear Medicinevol 46 no 1 2005

[7] J C Reubi J-C Schar B Waser et al ldquoAffinity profiles forhuman somatostatin receptor subtypes SST1-SST5 of somato-statin radiotracers selected for scintigraphic and radiotherapeu-tic userdquo European Journal of Nuclear Medicine vol 27 no 3 pp273ndash282 2000

[8] Y Menda M S OrsquoDorisio S Kao et al ldquoPhase I trial of 90Y-DOTATOC therapy in children and young adults with refrac-tory solid tumors that express somatostatin receptorsrdquo Jour-nal of Nuclear Medicine vol 51 no 10 pp 1524ndash1531 2010

[9] D J Kwekkeboom W H Bakker B L Kam et al ldquoTreat-ment of patients with gastro-entero-pancreatic (GEP) tumourswith the novel radiolabelled somatostatin analogue [177Lu-DOTA0Tyr3]octreotaterdquo European Journal of NuclearMedicineand Molecular Imaging vol 30 no 3 pp 417ndash422 2003

[10] D J Kwekkeboom W W de Herder B L Kam et alldquoTreatment with the radiolabeled somatostatin analog [177Lu-DOTA0Tyr3]octreotate toxicity efficacy and survivalrdquo Journalof Clinical Oncology vol 26 no 13 pp 2124ndash2130 2008

[11] J E Gains J B Bomanji N L Fersht et al ldquo177Lu-DOTATATEmolecular radiotherapy for childhood neuroblastomardquo Journalof Nuclear Medicine vol 52 no 7 pp 1041ndash1047 2011

[12] J RMasters J AThomson B Daly-Burns et al ldquoShort tandemrepeat profiling provides an international reference standardfor human cell linesrdquo Proceedings of the National Academy ofSciences of the United States of America vol 98 no 14 pp 8012ndash8017 2001

[13] D C Vines D E Green G Kudo and H Keller ldquoEvaluation ofmouse tail-vein injections both qualitatively and quantitativelyon small-animal PET tail scansrdquo Journal of Nuclear MedicineTechnology vol 39 no 4 pp 264ndash270 2011

[14] C B Johnbeck M M Jensen C H Nielsen A M F HagU Knigge and A Kjaer ldquo 18F-FDG and 18F-FLT-PET imagingfor monitoring everolimus effect on tumor-growth in neuroen-docrine tumors studies in human tumor xenografts in micerdquoPLoS ONE vol 9 no 3 Article ID e91387 2014

[15] K A Streby N Shah M A Ranalli A Kunkler and T P CripeldquoNothing but NET A review of norepinephrine transporter

expression and efficacy of 131I-mIBG therapyrdquo Pediatric Bloodand Cancer vol 62 no 1 pp 5ndash11 2015

[16] L-C Sun L V Mackey J Luo J A Fuselier and D H CoyldquoTargeted chemotherapy using a cytotoxic somatostatin con-jugate to inhibit tumor growth and metastasis in nude micerdquoClinical Medicine Oncology vol 2 pp 491ndash499 2008

[17] MMaggi E Baldi G Finetti et al ldquoIdentification characteriza-tion and biological activity of somatostatin receptors in humanneuroblastoma cell linesrdquo Cancer Research vol 54 no 1 pp124ndash133 1994

[18] C L Moertel J-C Reubi B S Scheithauer D J Schaid andL K Kvols ldquoExpression of somatostatin receptors in childhoodneuroblastomardquo American Journal of Clinical Pathology vol102 no 6 pp 752ndash756 1994

[19] C C Raggi MMaggi D Renzi et al ldquoQuantitative determina-tion of sst2 gene expression in neuroblastoma tumor predictspatient outcomerdquo Journal of Clinical Endocrinology and Meta-bolism vol 85 no 10 pp 3866ndash3873 2000

[20] A Al-Nahhas Z Win T Szyszko A Singh S Khan and DRubello ldquoWhat can gallium-68 PET add to receptor and mole-cular imagingrdquo European Journal of Nuclear Medicine andMolecular Imaging vol 34 no 12 pp 1897ndash1901 2007

[21] P F Rambaldi V Cuccurullo V Briganti and L Mansi ldquoThepresent and future role of 111119868n Pentetreotide in the PET erardquoQuarterly Journal of Nuclear Medicine and Molecular Imagingvol 49 no 3 pp 225ndash235 2005

[22] S G DuBois and K K Matthay ldquoRadiolabeled metaiodoben-zylguanidine for the treatment of neuroblastomardquo NuclearMedicine and Biology vol 35 no 1 pp S35ndashS48 2008

[23] J P Howard J M Maris L S Kersun et al ldquoTumor responseand toxicity with multiple infusions of high dose 131I-MIBG forrefractory neuroblastomardquo Pediatric Blood and Cancer vol 44no 3 pp 232ndash239 2005

[24] R J Hutchinson J C Sisson B Shapiro et al ldquo131-I-metaiodo-benzylguanidine treatment in patientswith refractory advancedneuroblastomardquo The American Journal of Clinical OncologyCancer Clinical Trials vol 15 no 3 pp 226ndash232 1992

[25] AQuigley J BuscombeGGopinathMCaplin andAHilsonldquoIn-vivo characterisation of the functional aspects of carcinoidtumors by imaging somatostatin receptors and amine uptakerdquoJournal of nuclear medicine p 74P 2003 SOC NUCLEARMEDICINE INC 1850 SAMUEL MORSE DR RESTON VA20190-5316 USA

[26] A Kroiss D Putzer C Uprimny et al ldquoFunctional imagingin phaeochromocytoma and neuroblastomawith 68Ga-DOTA-Tyr3-octreotide positron emission tomography and 123I-meta-iodobenzylguanidinerdquo European Journal of Nuclear Medicineand Molecular Imaging vol 38 no 5 pp 865ndash873 2011

[27] K P Koopmans P L Jager I P Kema M N Kerstens FAlbersy and R P F Dullaart ldquo111In-octreotide is superior to123I- metaiodobenzylguanidine for scintigraphic detection ofhead and neck paragangliomasrdquo Journal of Nuclear Medicinevol 49 no 8 pp 1232ndash1237 2008

[28] H Mohsin F Jia J N Bryan et al ldquoComparison of pretargetedand conventional CC49 radioimmunotherapy using 149Pm166Ho and 177Lurdquo Bioconjugate Chemistry vol 22 no 12 pp2444ndash2452 2011

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


macrocyclic chelator 14710-tetraazacyclododecane14710-tetraacetic acid (DOTA) DOTA-TATE is SSTR2 selectivewith a higher SSTR2 affinity (sim02 nM) in vitro comparingto two other commonly used somatostatin analogs [68Ga-DOTA0-Tyr3]octreotide (DOTA-TOC) and [DOTA01NaI3]octreotide (DOTA-NOC) [7] Peptide-receptor imag-ing and therapy with radiolabeled somatostatin analogsare an established and effective treatment option for adultpatients with SSTR-positive neuroendocrine tumors [4]DOTA-TOC and DOTA-TATE can also be radiolabeled with90Y or 177Lu for targeted 120573minus-particle radionuclide therapyof neuroendocrine tumors In a recent phase I trial thesafety and efficacy of 90Y-DOTA-TOC therapy were demon-strated in 17 children and young adults with refractory SSTR-positive neuroendocrine tumors including NB [8] The firststudy of 177Lu-DOTA-TATE treatment in 35 patients withgastroenteropancreatic neuroendocrine tumors was pub-lished in 2003 where an objective response of 38 wasachieved [9] In a 2008 evaluation of 310 adult patientswith neuroendocrine tumors an overall response of 30was reported [10] More recently a pilot clinical studydemonstrated that 68Ga-DOTA-TATE PET could be used toimage children with relapsed or primary refractory high-riskNB and 68Ga-DOTA-TATE PET could be used to identifypotential candidates for 177Lu-DOTA-TATE treatment [11]In this study 6 out of 8 children demonstrated high uptakeof 68Ga-DOTA-TATE and proceeded to treatment Patientsreceived 2 or 3 administrations of 177Lu-DOTA-TATE(03GBqkg 81mCikg per dose) at a median interval of 9weeks and a median administered activity of 73 GBq Fiveof these patients had the stable disease as assessed using theResponse Evaluation Criteria in Solid Tumors (RECIST)This study while limited in the number of patients studiedprovided proof-of-principle that children with NB can beimaged and treated with somatostatin receptor-targetedagents More interestingly this study demonstrated that 1patient (out of a series of 6 patients) whose disease wasnegative for 123I-MIBG nevertheless demonstrated markeduptake of 68Ga-DOTA-TATE

The primary purpose of this study is to evaluate theuptake of 68Ga-DOTA-TATE in NB xenograft models andcorrelate this uptake with the expression levels of SSTR2and therefore identify biomarkers which can predict thetherapeutic effects of 177Lu-DOTA-TATE

2 Materials and Methods

21 Materials and Reagents Gallium-68 and lutetium-177radiolabeled DOTA-TATE were supplied by the Centre forProbe Development and Commercialization (CPDC Hamil-ton ON Canada) 68Ga-DOTA-TATEwas produced with thespecific activity of 412 plusmn 99GBq120583mol and with the radio-chemical purity of gt97 at all cases NET antibody (NET17-1) was purchased from MAb Technologies (Stone MountainGA) and SSTR2 antibody (ab134152) was obtained fromAbcam (Cambridge MA) Triton X-100 ethylenediaminete-traacetic acid (EDTA) and sodium dodecyl sulfate (SDS)

were purchased from Sigma Chemical Company (St LouisMO)

22 Cells and Cell Culture Eight NB cell lines (NUB-7 SK-N-BE(2) BE(2)C LAN-5 SH-SY5Y CHLA-15 CHLA-20and CHLA-90) were selected to represent a panel of celllines with different biological and genetic backgrounds ofNB (Table 1) NUB-7 LAN-5 SK-N-BE(2) BE(2)C andSH-SY5Y neuroblastoma cells were kindly provided by DrHerman Yeger (The Hospital for Sick Children TorontoON Canada) CHLA-15 CHLA-20 and CHLA-90 wereobtained from the Childrenrsquos Oncology Group Cell Cultureand Xenograft Repository (httpwwwcogcellorg) under asigned and approved Material Transfer Agreement Cell lineauthentication was performed using short tandem repeats(STR) DNA profiling (Promegarsquos GenePrint 10 System) [12]conducted by the Genetic Analysis Facility at the Centrefor Applied Genomics of The Hospital for Sick ChildrenThe DNA (STR) profile for all cell lines was matched tothe profile listed in the Childrenrsquos Oncology Group (COG)STR Database (httpstrdbcogcellorg) CHLA-15 CHLA-20 and CHLA-90 neuroblastoma cells were cultured inIscoversquos modified Dulbeccorsquos medium supplemented with3mML-glutamine 5120583gmL insulin 5120583gmL transferrin and5 ngmL selenous acid (ITS Culture Supplement Collabo-rative Biomedical Products Bedford MA) and 20 fetalbovine serum (FBS) NUB-7 LAN-5 SK-N-BE(2) BE(2)Cand SH-SY5Y neuroblastoma cells were cultured in 120572-MEMsupplemented with 10 FBS

23 RT-PCR Total cellular RNA was prepared using Qia-gen RNeasy mini kit (Qiagen Valencia CA) according tothe manufacturersrsquo instruction Residual DNA was elim-inated using the Qiagen RNase-Free DNase Set cDNAswere synthesized from 2 120583g of RNA with the SuperscriptII Reverse Transcriptase (Invitrogen) PCR was performedusing 1120583L of cDNA in the PCR buffer supplemented with02mM of dNTP 25 units of Taq polymerase (Biorad andThermoFisher Scientific) and 05 120583M of each sense andantisense primer The following primers were used SSTR2forward 51015840-GGTGAAGTCCTCTGGAATCC-31015840 and reverse51015840-CCATTGCCAGTAGACAGAGC-31015840 NET forward 51015840-CTCAAGGAGGCCACGGTATGGATCG-31015840 and reverse 51015840-ACCTGGAAGTCATCAGCCAGTCCGG-31015840 GAPDH for-ward 51015840-CTGTCCAGTTAATTTCTGACC-31015840 and reverse 51015840-CTTTGTACATGGTATTCACCAC-31015840 PCR products wererun on a 15 agarose (Invitrogen) with a 100 bp marker(Thermo Fisher Scientific) and stained with ethidium bro-mide Gel pictures were taken using the AlphaImager 2200(Alpha Innotech Kasendorf Germany)

24 Western Blot Theprotein lysates were analyzed byWest-ern blot for SSTR2 and norepinephrine transporter (NET)Briefly cells were lysed in lysis buffer and denatured Sampleswere separated using 10 Bis-Tris precast gels (Invitrogen)followed by transfer to a PDVFmembrane After blocking allmembranes were incubated overnight at 4∘C in TBST (Tris-buffered saline 01Tween 20) buffer containing the primaryantibodies Primary antibody complexes were then detected




















3 Results



SSTR2

NET

GAPDH

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(a)

SSTR2

NET

-Actin

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(b)










(a)


(b)10

08

06

04

02

00

Tum

or S

U６

Ｇ

Ｈ

lowastlowast

CHLA-15 SK-N-BE(2)

(c)



4 Discussion



SK-N-BE(2)

(b)

CHLA-15

(a)


(c) (d) (e)

2mm


0 10 15 200

1

2

3

4


Tum

or v

olum

e (c Ｇ

3)


lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t


5


(b)




(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































3 Results



SSTR2

NET

GAPDH

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(a)

SSTR2

NET

-Actin

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(b)










(a)


(b)10

08

06

04

02

00

Tum

or S

U６

Ｇ

Ｈ

lowastlowast

CHLA-15 SK-N-BE(2)

(c)



4 Discussion



SK-N-BE(2)

(b)

CHLA-15

(a)


(c) (d) (e)

2mm


0 10 15 200

1

2

3

4


Tum

or v

olum

e (c Ｇ

3)


lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t


5


(b)




(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SSTR2

NET

GAPDH

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(a)

SSTR2

NET

-Actin

CHLA

-15

CHLA

-20

CHLA

-90

LAN

-5

SK-N

-BE(

2)

BE(2

)C

NU

B-7

SH-S

Y5Y

(b)










(a)


(b)10

08

06

04

02

00

Tum

or S

U６

Ｇ

Ｈ

lowastlowast

CHLA-15 SK-N-BE(2)

(c)



4 Discussion



SK-N-BE(2)

(b)

CHLA-15

(a)


(c) (d) (e)

2mm


0 10 15 200

1

2

3

4


Tum

or v

olum

e (c Ｇ

3)


lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t


5


(b)




(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)10

08

06

04

02

00

Tum

or S

U６

Ｇ

Ｈ

lowastlowast

CHLA-15 SK-N-BE(2)

(c)



4 Discussion



SK-N-BE(2)

(b)

CHLA-15

(a)


(c) (d) (e)

2mm


0 10 15 200

1

2

3

4


Tum

or v

olum

e (c Ｇ

3)


lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t


5


(b)




(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SK-N-BE(2)

(b)

CHLA-15

(a)


(c) (d) (e)

2mm


0 10 15 200

1

2

3

4


Tum

or v

olum

e (c Ｇ

3)


lowast

lowast

(a)

0

50

100

o

f orig

inal

bod

y w

eigh

t


5


(b)




(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

0

2

4

6

8

10


）123-MIBG SPECT

lowastlowast

lowastlowast

Tum

orm

uscle

ratio

(b)

0

1

2

3

4


lowast

lowast

lowast

lowast


Tum

orm

uscle

ratio

(c)

SK-N-BE(2)1 2 3 1 2 3 1 2 3

CHLA-15 BE(2)C

NET

-Actin

(d)

00

05

10

15

NET

expr

essio

n


(e)







5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions





Acknowledgments



References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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